This project involves the role of two related substrates for protein kinase C (PKC) in mediating the many cellular effects resulting from activation of this family of kinases by hormones, neurotransmitters and other agonists. The PKC substrates under study are MARCKS and its smaller related protein, the MARCKS-like protein 1, or MARCKSL1. Ongoing projects include structure-function studies of the protein and its mutant derivatives in the development of the mouse central nervous system. These studies have involved creating gene knockouts for MARCKS and MARCKSL1 in the mouse. In addition, transgenic complementation of the knockout mice with mutant proteins is continuing in order to analyze structure-function relationships in development. Similar studies are being performed in a cell transfection system, which seeks to examine the effect of the wild-type and mutant proteins on cellular adhesion and migration on various matrices. A novel aspect of this project involves the development of ES cell lines in which MARCKS and MARCKSL1 have been knocked out, and which also express an immunohistochemical marker such as beta-galactosidase. These are being used to determine the cell-specific or substrate-specific nature of the knockout phenotype, by using the ES cells to create chimeric mice. Finally, we are currently investigating the possibility that mutations in the MARCKS and MARCKSL1 genes are involved in human neural tube defects, particularly at the level of increasing a genetic predisposition to environmental causes of these defects. These studies have recently been expanded to include studies of another protein factor that is critical in brain development, RFX4_v3. This winged helix transcription factor is important for normal brain development, as indicated by gene disruption experiments showing that null mutants for this gene have major developmental brain defects that are incompatible with life, and that mice heterozygous for this mutation develop congenital non-communicating hydrocephalus. These studies are being expanded into the evaluation of the promoter activity that leads to expression of this transcription factor;the identification of target genes and their expressed proteins;and the identification of protein co-activators. The studies are also being extended into human populations with non X-linked congenital hydrocephalus, whose genetic causes and environmental predisposing factors are largely unknown.